To what extent does the Australian and Malaysian originated carrots (Daucus carota) affect the concentrations of β-carotene present by extracting β-carotene from the Daucus carota from using solvent extraction and measuring the absorbance of β-carotene using a UV Spectrophotometer, followed by calculating the concentration of β-carotene present in both origins of Daucus carota?
The consumption of vegetables over the years has been increasing tremendously due to the myriad of people changing their lifestyles in order to lead healthier lives. Furthermore, the consumption of organically-produced foods has been drastically increasing due to the attention it obtains from social media. Thus, there is a paradigm shift in terms of food choices as now, more people are consuming healthier foods. According to a survey done by The Research Institute of Organic Agriculture and the International Federation of Organic Agriculture Movements in 2015, it showed that the growth in the market for organic foods (vegetables) had increased almost five-fold since 1999 [15.2 bn $USD] to 2013 [72.0 bn $USD] (Willer). This statistic supports the fact that the consumption of vegetables is ever growing.
Australian farms predominantly grow their vegetables organically. This means that about 25 pesticides are only allowed to be used to produce organic foods as compared to conventional production in Malaysia where over 900 pesticides can be used (Roseboro). Additionally, This is also another contributing factor to why so many people prefer organically-produced food over conventionally grown foods as it is a healthier option. However, the price allocated to organically-produced foods is considerably higher than conventionally grown foods. I wanted to see whether the concentration β-carotene correlated with the origin of the Daucus carota (CABI) itself. Moreover, I wanted to know why my parents would rather buy carrots from a specific origin such as Australia, even when given the high price?
β-carotene is the orange pigment which is found in photosynthetic organisms. It is instrumental in photosynthesis, and is an accessory pigment in the light-dependent reaction. It is also an antenna pigment due to its properties, enabling it to form protein complexes such as photosystems that absorb photons of light (Andrew Allot). β-carotene’s main function is to essentially defend the plant from molecular oxidation from singlet oxygen which are produced from chlorophyll triplet states. Singlet oxygen is a highly unstable molecule produced during photosynthesis and can lead to oxidation or isomerisation of the plant. Oxidation or isomerisation could hinder the process of photosynthesis in the plant.
UV-Spectrophotometry will be used to determine the concentration of β-carotene present in the Daucus carota. It essentially measures the absorbance of a particular solution in order to determine the concentration of β-carotene. Based on the absorption spectrum of β-carotene, β-carotene absorbs the most light around 410nm-490nm(Evens). Hence, the colour region would be calibrated to the green-blue colour region according to the respective wavelength chosen, in this case being 450nm. Once the absorbance of β-carotene is determined, the concentration of β-carotene can be calculated using
\(\frac{A×V(ml)\times10^4}{A^{1\%}_ {1cm}\times \ P\ (g)}\)
A refers the absorbance used [450nm], V refers to the volume of β-carotene extracted. P refers to the weight of Daucus carota used and \(A^{1\%}_ {1cm}\) represents the β-carotene coefficient [2500] (Cucurbita Moschata Duch).
Organic farming uses a higher concentration of potassium/K^{+} ions as compared to conventional farming methods (Fess ID, Benedito). The increase in K^{+} ions correlates with the increase in ATP [Adenine tri-phosphate] formation during aerobic respiration in the plant. More ATP corresponds to a higher rate of hypertrophy, which increases the size of the organelles. Therefore, having an enlarged cell would allow for a higher concentration of β-carotene to occupy the thylakoid membranes of the chloroplasts (Bogacz-Radomska, Harasym). Furthermore, a study indicated that photosynthetic rate can also be affected by the concentration of metal ions present in the air (Tarek Houri, Yara Khirallah, et al). This would lead to the decline in the concentration of β-carotene as metal ions and primary pollutants attack the chloroplast, leading to the destruction of its organelles (Sewelam, et al). Since β-carotene is located in the thylakoid membrane, it would also be adversely affected. Metal pollution is determined to be higher in Malaysia than in Australia (Bernhard A, et al), (Laidlaw, et al). Therefore, this leads to the hypothesis.
H_{1}:The Australian Daucus carota will contain a higher concentration of β-carotene as compared to Malaysian Daucus carota.
H_{0}: There is no statistical significance between the concentrations of β-carotene between the Malaysian and Australian originated Daucus Carota.
25 cm^{3}
using a25 cm^{3}
measuring cylinder\(\bigg(±0.5cm^3\bigg)\)
50 cm^{3}
± 1 cm^{3}
50.0 cm^{3}
± 0.5 cm^{3}
500 cm^{3}
250 cm^{3}
25.0 cm^{3}
± 0.5 cm^{3}
100 cm^{3}
± 1 cm^{3}
500 cm^{3}
1100 cm^{3}
2000 cm^{3}
2000 cm^{3}
\(\bigg(\frac{1}{100} + \frac{0.01}{10}\bigg)\times100{\%}\\= ± 1.1\%\)
In order to obtain the concentration of the β-carotene, the process will be needed to be split into 3 separate sections. The first process requires the extraction of carotene from the carrot. This is followed by measuring the absorbance of the carotene. Finally, the absorbance of all the values will be used to calculate the concentration of β-carotene. A t-test is then used in order to evaluate the accuracy of the data (Rodriguez-Amaya).
concentration of β-carotene,\(\frac{A×V(ml)\times10^4}{A^{1\%}_{1cm} × P \ (g)}\).
Where A: Absorbance reading found at 450nm
V: Total extracted volume of β-carotene in mL
\(A^{1\%}_{1cm}\) β-carotene coefficient at 2500
P: Sample weight in grams
A T-test will also be used. This is to determine the statistical significance as well as determine whether there is a significant difference in terms of their β-carotene concentrations.
The formula is: t = \(\frac{(x_1-x_2)}{\sqrt{\frac{(s_1)^2}{n_1}+\frac{(s_2)^2}{n_2}}}.\) x_{1} and x_{2} is the mean of the sample sizes of each origin of Daucus carota(Australian and Malaysian). s_{1} and s_{2} refer to the standard deviation of each sample while n is the sample size of each origin.
Sample Calculation: [Trial 1 of Australian originated Daucus carota] To determine the concentration of β-carotene in 10.00 grams of Daucus carota.
Using the equation,\(\frac{A\times V(ml)\times10^4}{A^{1\%}_{1cm}\times P\ (g)}.\)
Where A: Absorbance reading found at 450nm
V: Total extracted volume of β-carotene in mL
\(A^{1\%}_{1cm}\) β-carotene coefficient at 2500
P: Sample weight in grams
= 0.00034566 g
≈0.000346 g
Concentration of β-carotene = \(\frac{0.000344566}{(\frac{10}{1000})}\)
= 0.035 gdm^{-3}
^{T-test to determine statistical difference between differing originated Daucus Carota}
^{t = \(\frac{(0.036390-0.0313215)}{\sqrt{\frac{(0.003)^2}{10}+\frac{(0.005)^2}{10}}}\)}
t = 8.2535
To determine the degrees of freedom(df) for t-test:
(Sum of sample size of both origins) – 2
= 18
\(\frac{0.01}{10}\times100\)
25.0 cm^{3}
Measuring Cylinder25.0 cm^{3}
±0.5 cm^{3}
\(\frac{0.5}{25}\times100\)
25.0 cm^{3}
Measuring Cylinder25.0 cm^{3}
±0.5 cm^{3}
\(\frac{0.5}{25}\times100\)
50.0 cm^{3}
Measuring Cylinder30.0 cm^{3}
±0.5 cm^{3}
\(\frac{0.5}{30}\times100\)
\(\frac{0.01}{10}\times100\)
100 cm^{3}
Measuring Cylinder100 cm^{3}
±1 cm^{3}
\(\frac{1}{100}\times100\)
100 cm^{3}
Measuring Cylinder100 cm^{3}
±1 cm^{3}
\(\frac{1}{100}\times100\)
\(\frac{0.005}{2.599}\times100\)
\(\bigg(\frac{0.01}{10}+\frac{0.5}{25}+\frac{0.5}{25}+\frac{0.5}{30}+\frac{0.01}{10}+\frac{1}{100}+\frac{1}{100}+\frac{0.005}{2.599}\bigg)\)×100
=8.058%
≈8.06%
The figure above shows a bar graph indicating the concentration of -carotene against the origin of Daucus carota. From the bar chart, the higher mean concentration of β-carotene is from the Australian originated Daucus carota of 0.036 gdm^{-3}, while the Malaysian originated Daucus carota had a lower mean β-carotene concentration of 0.031 gdm^{-3}. Therefore, Australian originated Daucus carota have a higher β-carotene concentration than Malaysian originated Daucus carota. Furthermore, Australian originated Daucus carota had a higher mean absorbance value at 2.599 ABS than Malaysian originated carrots with a mean absorbance of 2.238 ABS. From this, we can conclude that Australian originated carrots contained a higher concentration of β-carotene as compared to Malaysian originated carrots. In terms of their standard deviation, the standard deviation of the Australian originated carrots is lower at 0.002815 as compared to Malaysian originated carrots with a standard deviation of 0.005453. This indicates that Malaysian originated Daucus carota concentrations have a wider spread. Therefore, it is less reliable and this makes the experiment less precise, as the Malaysian originated Daucus carota’s concentration is spread over a wider range. After the 33% of the mean and the standard deviation was calculated, the standard deviation was lower than the 33% mean. This indicates that the data is reliable. Additionally, based on the graph, the error bars that were present were large and this indicated that there was a weak correlation. Additionally, the data collected was more variable from the mean itself. In terms of the data’s significance, it could not be deduced from the graph itself, as a T-test would need to be conducted. However, the error bars overlapped. This could indicate that the difference in concentrations between the Australian and Malaysian Daucus carota were not significant. However, this cannot be confirmed until a T-test is carried out. Therefore, the size of the error bars did not make the conclusion that Australian originated Daucus carota has a higher concentration of β-carotene than Malaysian originated Daucus carota difficult to reach. Moving on to the t-test, the degrees of freedom [d_{f}] was determined to be 18.
Furthermore, in Biology, the significance value is usually taken as 5% [0.05]. From the table, it was determined that the critical T-value was 2.101. The calculated T-value was determined to be 8.255. Since the calculated T-value was higher than the critical T-value, it indicated that the difference between the mean concentrations of Australian and Malaysian originated Daucus carota does have a significant statistical difference. This contradicts with the error bars in the graph, as from the graph it seems as if the difference in their respective concentrations are not statistically significant. However, from the t-test, the conclusion is drawn that there is in fact a statistical difference between the concentrations of Australian and Malaysian Daucus carota.
Going back to my Research Question, “To what extent does the Australian and Malaysian originated carrots (Daucus carota) affect the concentrations of β-carotene present by extracting β-carotene from the Daucus carota from both origins using a separating funnel and measuring its absorbance using a UV Spectrophotometer, followed by calculating the concentration of β-carotene present in both Daucus carota?”. In conclusion, the Australian originated Daucus carota indeed did have a higher concentration of β-carotene that the Malaysian originated Daucus carota. This supports the H_{1}_{ }hypothesis, which states that Australian originated Daucus Carota would possess a higher concentration of β-carotene as it possesses a lower concentration of primary pollutants as well as a higher concentration of K^{+}^{ }ions in the soil. Moreover, my data rejects the null hypothesis [H^{0}], which states that there would be no statistical significance between the difference in the concentrations of β-carotene between the Australian and Malaysian Daucus carota. The evidence supporting the hypothesis is seen in Graph 1, where it shows that the Australian originated Daucus carota ccontains a higher mean β-carotene concentration (0.036390 gdm^{-3}) than the Malaysian originated Daucus Carota (0.031325 gdm^{-3}^{}). The null hypothesis was contradicted by a t-test which showed that the calculated T-value (8.255) was higher than the critical T-value (2.101), which showed that there was a significant statistical difference in terms of the different concentrations of β-carotene. The differing concentrations of β-carotene does answer our research question, by showing that differing origins does affect the concentration of β-carotene present in the Daucus carota due to external factors affecting the production of β-caroten such as the quality of soil and the concentration of primary pollutants in the atmosphere. Although there is not any scientific data published that specifically shows how Australian and Malaysian originated Daucus carota affect the concentrations of β-carotene. The absorbance readings were collected at 450nm as usually, carotenoids are measured at λmax ranging from 410nm to 490nm (ResearchGate). This indicates that the wavelength in which β-carotene i Thus, I had decided to take the average wavelength, being 450nm, where the absorption of β-carotene would be the maximum. The percentage error was calculated by
\(\frac{[Experimental\ Value-Literature\ Value]}{Literature\ Value}\)×100
There was no specific literature value regarding the concentration of β-carotene in the Australian and Malaysian originated Daucus carota. However, an article (ResearchGate) had indicated that the concentration of β-carotene ranged from 3.20 mg/kg all the way to 170.00 mg/kg (Tanveer Ahmad). Thus the average was taken in order to calculate the percentage error. Since our experiment determined the concentration in grams, the concentration of β-carotene was multiplied by 1000. This made the average concentration of β-carotene 36.40 g/kg in Australian originated carrots and 31.30 g/kg in Malaysian originated carrots. The percentage error was determined to be -56.4% and -62.5% in Australian and Malaysian originated carrots respectively. The negative value indicates that the experimental value was lower than the literature value. The average percentage uncertainty of the experiment was also calculated to be 8.06% and 8.09% for the Australian and Malaysian originated Daucus carota as seen in the sample calculation for the Australian originated Daucus carota. The difference in the percentage uncertainties is due to the lower average absorbances observed in Malaysian originated carrots. Causing the increase in percentage uncertainty. Since the percentage error is higher than the percentage uncertainty, it indicates that systematic errors were tantamount in this experiment as compared to random errors.
The experiment did possess a very low percentage uncertainty of about 8%, which was calculated from the sample calculation. Furthermore, the error bars on the graph indicates a lower possibility of error occurring. Additionally, the concentration β-carotene was only obtained from the middle of the storage root of the Daucus carota. This was to prevent any uncertainty, as different parts of the carrot may contain different concentrations of β-carotene, thus affecting the accuracy of the data. Moreover, the experiment was conducted rapidly. This is to prevent the oxidation or isomerisation of the carotenoids. Another strength was how the experiment was adapted in order to yield the most volume of β-carotene with wasting the least volume of reagents. At first, the volume of hexane used was 5 cm^{3} . However, this did not yield a sufficient volume of β-carotene as it would not be enough to fill up a cuvette. On the other hand, the volume of hexane used was 50 cm^{3}. This yielded too much β-carotene which meant that a large volume of hexane and acetone was wasted. And this contradicted with one of the ethical considerations. Thus, the volume had been adjusted to 25 cm^{3}. Furthermore, β-carotene is susceptible to changes in heat, light intensity, humidity as well as molecular oxygen. Since the experiment was carried out in an open environment, it could cause the β-carotene go oxidise and could even isomerase (Yahia). This would cause the concentration of β-carotene to deteriorate. This affects the precision of the data, as this would mean that the concentration of β-carotene would always be different even if it were taken from the same part of the Daucus carota. Therefore, the experiment was planned out first and the experiment was conducted in a darker environment, such as a fume cupboard. This would prevent light causing any decomposition of the β-carotene.
Instead of having the origin of Daucus carota as the independent variable, the effect of temperature on the concentration of β-carotene can also be investigated.. Additionally, the deterioration of β-carotene can also be investigated. This will be achieved by measuring the initial concentration of β-carotene, followed by heating it to different temperatures and calculating the concentration of β-carotene after heating.
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